One of the fundamental tasks of basic retina research is to understand how the retina - a thin sheet of light- sensitive tissue at the back of te eye - is responsible for processing and transmitting signals arising from the absorption of photons. This process begins at the very first synapse in the retina, which transmits signals from rod and cone photoreceptors to second-order neurons. The signaling capabilities of photoreceptors depend the synaptic ribbon, a specialized structure found in a variety of neurosensory cells responsible for providing a supply of primed vesicles to support signaling by tonic vesicle release. The objective of this proposal is to explore the fundamental signaling capabilities of the cone ribbon synapse by examining how synaptic ribbons are replenished with vesicles and how that replenishment mechanism contributes to early stages of visual processing in the retina. Previous studies have described the ways in which retinal ribbon synapses are capable of signaling contrast and luminance in a manner dependent on the kinetics of synaptic vesicle replenishment. Recent work in the Thoreson lab has shown that the kinetics of replenishment at the cone ribbon is also accelerated by calcium (Ca2+), pointing to a Ca2+-dependent mechanism for regulating the encoding of photoreceptor signals. At the Calyx of Held, an auditory relay synapse, acceleration of replenishment by calcium depends on the signaling molecule calmodulin. Calmodulin may similarly regulate the Ca2+- dependence of replenishment at photoreceptor synapses, but this possibility has yet to be tested. Aim 1 will test the hypothesis that calmodulin is responsible for a fast, Ca2+-dependent mechanism of vesicle replenishment at the cone ribbon. Additionally, replenishment has been suggested as a major determinant of kinetic-encoding responses by the photoreceptor synapse, yet this also remains untested. Aim 2 will test the hypothesis that a fast calmodulin-dependent mode of replenishment is responsible for encoding and transmitting the timing of visual responses. These goals will be accomplished using a variety of electrophysiological techniques such as electroretinogram and single and paired whole-cell recordings as well as with live imaging techniques such as confocal calcium imaging, TIRF microscopy, and single-particle tracking with quantum dots to assess synaptic signaling by cones. Specific pharmacological compounds, delivered directly to the photoreceptor or applied to the entire retina, will be used to manipulate signaling by calmodulin and Ca2+-dependent processes. Proper encoding and transmission of signals by photoreceptors is crucial to vision. Understanding retinal function in health as well as disease is important when implementing therapeutic approaches that integrate with the existing retinal network such as stem cells or retinal implants. Moreover, an understanding of the mechanisms regulating vesicle resupply and trafficking in photoreceptors, as this proposal seeks to provide, is important in understanding the pathophysiology of several retinal degenerative diseases, as synaptic proteins with roles in the neurotransmission have been implicated in various retinopathies.